Proteins are important components of cells and their activities determine various cellular functions. Abnormal protein expression and activity may cause cell to malfunction. Although an organism' genome may encode tens of thousands of proteins, a given cell of the organism usually expresses only a fraction of the proteins; and the protein expression pattern determines a cell's shape and function. In biomedical research, it is usually desirable to know what proteins are expressed in a cell under a specific condition. By comparing protein expression profiles, it is possible to identify those proteins whose expressions and activations are responsible for the differences between cell types.
Diseases alter protein expressions and abnormal protein expressions are the causes of many diseases. Therefore, determination and comparison of the expression profiles between normal and abnormal biological samples are useful for understanding disease mechanisms. Simultaneous detection of multiple proteins is also useful in clinical diagnostics. For example, examining several viral proteins is more reliable than examining just one viral protein for the diagnosis of viral infection. Profiling protein expressions is also valuable in distinguishing normal cells from early-stage cancer cells, and from metastatic cancer cells. In addition, protein expression profiling is useful in key areas of drug development, such as in target selection, toxicology, and the identification of surrogate markers for drug response.
A protein with a specific amino acid sequence may be present in different isoforms due to posttranslational modifications. There are many types of protein posttranslational modifications including phosphorylation, glycosylation, lipidation, and ubiquitination. They play important roles in regulating protein activities and functions. Phosphorylation at serine, threonine or tyrosine residues is an important mechanism in signal transduction. Aberrant protein phosphorylation contributes to many human diseases. In a cellular process, certain proteins are characteristically modified and activated. Detection of these modified proteins can provide valuable information on that cellular process. Among the methods of detecting protein phosphorylations, metabolic labeling with radioisotopes and immuno-detection with antibodies against phosphoproteins are most commonly used. However, these methods are only applicable to the analysis of one or a few proteins at a time. Although antibodies specific for phosphorylated amino acids (e.g., PY20, 4G10) can reveal multiple phosphorylated proteins, they alone are unable to identify individual phosphorylated proteins. New methods for simultaneous detection of multiple phosphorylated or other modified proteins are highly desirable for signal transduction studies and clinical diagnosis.
It has long been the goal of molecular biologists to develop technologies that can reliably quantify the expressions of every protein and its isoforms in a biological sample. However, this has turned out to be difficult to achieve. Traditionally, the expression of one or a small number of proteins can be detected by immunological methods, such as Western blotting and Enzyme-Linked Immunosorbent Assay (ELISA). Western blotting (Immunoblotting) is a widely used technique in protein research. It combines the resolution of gel electrophoresis with the specificity of immunochemical detection and is powerful in determining a number of important characteristics of protein antigens, e.g., the relative molecular weight and the quantity of an antigen in a protein sample. When combined with immunoprecipitation, Western blotting allows very sensitive detection of low-abundant antigens and more importantly, the specific interactions between proteins. It is also useful in detecting protein posttranslational modifications, e.g., protein tyrosine phosphorylation (Kamps, 1991).
A standard procedure for Western blotting includes the steps of separating proteins by gel electrophoresis, transferring proteins from a gel to a membrane support, and detecting an antigen with its specific antibodies. Electrophoretic separation of proteins is usually performed in polyacrylamide gels. These gels are usually cast between a pair of glass plates by polymerizing a solution of acrylamide monomers into polyacrylamide chains and simultaneously cross-linking the chains into a semisolid matrix. The pore size of a gel can be varied by adjusting the concentrations of polyacrylamide and the cross-linking reagent. When a mixture of proteins is applied to a gel and an electric current is applied, smaller proteins migrate faster than larger proteins through the gel. Therefore, proteins are separated (distributed) in the gels according to their molecular weights.
Several strategies have been used to increase the throughput of Western blotting. In one method, a protein blot is probed with a mixture of two or more antibodies which bind to proteins at different molecular weight positions on the blot. The antibodies preferably contain no cross-reactivity so that the signal generated by each antibody can be easily identified. In another method, a blot can be sequentially probed with multiple antibodies. That is, after a blot is probed with an antibody, the antibody is stripped off the blot. Then, a different antibody is applied to the blot. In a third method, the previously probed antibody can be left on the blot but its ability to generate signals is blocked before a new antibody is applied (Krajewski, Zapata, and Reed, 1996. Analytical Biochemistry 236, 221-228).
Another strategy to increase the efficiency of Western blotting is to use a device to facilitate the probing of multiple proteins on the same protein blot. Such a device contains multiple compartments. When assembled with a protein blot, the device divides the blot into many separate locations, each being enclosed by a compartment. Antibodies are applied to the compartments, each to a different one so that it reacts with its specific antigen at a blot location enclosed by the compartment. One such device is Multiscreen Blotting apparatus from Bio-Rad (Hercules, Calif.). The apparatus comprises two major parts: a base plate and a sample plate. The sample plate contains many channels/compartments arranged in parallel columns. Each column has one channel, which is open at one side and closed at the other side. The closed side has two outlets through which solutions can be introduced to and removed from the channel. When a protein blot is placed between the base plate and the sample plate, the channels divide the blot into multiple leak-proof, enclosed areas. Antibody solution introduced into a channel will only react with the antigens enclosed by that channel. When a plurality of antibodies are applied to the channels, each to a different channel, multiple antigens can be detected on the sample blot, each at a specific location enclosed by a channel.
The channels in the Multiscreen Blotting apparatus are arranged vertically with each channel spanning a full vertical position. When assembled with a protein blot formed by transferring proteins from one-dimensional SDS-polyacrymide gel, each channel covers proteins with a full-range of molecular weights. Multiple Blotting apparatus is not suitable for the protein blots formed by transferring proteins from two-dimensional gels. The number of channels in the current device is small, up to a few dozens. Multiscreen blotting apparatus from Bio-Rad has 20 channels. The current device is usually used with a protein blot transferred from a curtain SDS-polyacrylamide gel. Therefore, only one protein sample can be analyzed in each blot. Another disadvantage of the current method is that antibodies have to be introduced to each channel individually. This becomes cumbersome when a large number of antibodies are used.
Many proteome analyses are carried out with two-dimensional (2D) gel electrophoresis for protein separation followed by mass spectrometry for protein identification. 2D gel electrophoresis requires more complicated procedures than one-dimensional gel electophoresis and it is necessary to determine the identities of the proteins displayed on the two-dimensional gel, which is difficult to achieve for many proteins especially lower-abundance, basic and membrane proteins. Furthermore, despite the high resolving capability of 2D gel, multiple proteins are frequently found in one 2D gel spot so that quantitative and comparative analysis of 2D gel is prone to mistake. Protein identification by mass spectrometry-based technologies has improved significantly in recent years with the introduction of matrix-assisted laser-desorption ionisation/time-of-flight and electrospray ionisation methods.
Partly encouraged by the success of DNA microarrays in profiling mRNA expressions, strategies have been developed to use protein arrays in parallel examination of proteins. Protein arrays have been used for examining protein expression, protein phosphorylation, protein-protein interaction, protein-DNA interaction, and protein-analyte interaction (Lueking, et al. 1999. Protein microarrays for gene expression and antibody screening. Anal. Biochem. 270, 103-111. Ge, 2000 UPA, a universal protein array system for quantitative detection of protein-protein, protein-DNA, protein-RNA and protein-analyte interactions. Nucleic. Acid Research. 28, e3. Zhu, H. et al., 2001 Global analysis of protein activities using proteome chips. Science 293, 2101-2105. Wang et al. 2000 Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB activation. Mol. Cell. Biol. 20, 4505). However, there are several difficulties that prevent protein arrays from becoming a quantitative tool. Notably, due to the non-specific binding of capture reagents (e.g. antibodies), protein arrays lack specificity in examining protein expressions. Additional protein identification criteria are needed to increase assay specificity.
Other methods have also been used for examining protein expressions and functions. Immunochemical staining is a versatile technique in determining both the presence and localization of an antigen (Harlow and Lane, Antibodies, a laboratory manual, Cold Spring Harbor Press, 1988). An antibody array-based staining method was also developed for examining protein expression, protein cellular and subcellular localization, and other protein properties (Wang, 2004, Immunostaining with dissociable antibody microarrays. Proteomics 4, 20-26).